Human Methionine Sulfoxide Recuctase Can Inhibit Fragmentation Of Apoliporprotein B-100 In Cu～(2+) Oxidized Low Density Lipoprotein

The main objective of our study was to investigate the effect of human Methionine Sulfoxide reductase A ( hMSRA) antioxidant potential upon inhibition of fragmentation of apolipoprotein B-100 (apo B-100), that is the base for change of oxidized Low Density Lipoprotein (oxi. LDL) receptor-ligand from cellular uptake into macrophage uptake the first step in the development of atherosclerosis.At first, in order to get a large amount of hMSRA, we transferred PQE 6Xhis tagged hMSRA plasmid into E.coli Ml 5 (pREP4) strains, then extracting the cloned plasmid, and checked by restrictive enzymes digestion. After induction by adding IPTG in the culturing antibiotic LB medium, purified hMSRA protein was obtained by affinity chromatography using by Ni-NTA agarose column from the cloned E.coli bacteria and assessed by SDS-PAGE (its molecular weight), the activity of purified enzyme was examined by Trx/NTR/NADPH system and Western blotting( its antigen), and the protein concentration was determined by the Brad -Ford assay.On the next step, we collected freshly human plasma from healthy individuals, and separated LDL, with a density range of 1.020 -1.063 g/ml, using polyanions precipitation and short-time density gradient ultracentrifugation. Purified LDL was dialyzed against PBS solution, and oxidized by Cu2+ (5, l0mol/L). The anti-oxidative effect of hMSRA was assessed by relative electrophoresis mobility on Lipoprotein agarose gel for LDL, and on gradient SDS-PAGE for apo B-100 fragmentation. In addition, the effect of hMSRA on lipid peroxidation was assessed by thiobarbituric acid reactive, substances (TBARS) method. The results indicated that hMSRA inhibited fragmentation of apo B-100 in the oxidized LDL, and this potential of hMSRA is the same even if it added pre-, together, or post-oxidation of LDL by Cu2+. On the other hand, although hMSRA has a potential in reducing oxidized LDL, this potential was so high if it added pre-or together with Cu2+ rather than the beginning of oxidation. In related to prevention of lipid peroxidation production, eventhough, hMSRA has a potential, this potential decreases when hMSRA concentration increase in the sample. This is in opposite to that of the lipid peroxidation inhibition characteristic of Vit. C, that is, the potential increases with increase concentration of Vit. C. This finding indicates, the potential of hMSRA inhibition of LDL oxidation is different from prevention of production of lipid peroxidation.In general, in the present study, by expression and purification hMSRA in large scale from E.coli Ml5 strain, we demonstrate at the first time that hMSRA has a potential to inhibit fragmentation of apo B-100 in the Cu 2+ oxi. LDL, and may protect LDL against oxidation.